Cyclodextrins, also known as Schardinger dextrins, are starch-derived cyclic compounds containing six, seven, or eight .alpha.-D-glucopyranose residues linked by .alpha.-1,4 bonds. They are known as .alpha.-, .beta.-, .gamma.-cyclodextrin depending on the number of glucose residues, 6, 7 or 8, respectively. These cyclized molecules have neither a non-reducing nor reducing end-group. The cyclic nature of these compounds produces a cavity which is predominantly hydrophobic enabling the formation of inclusion complexes with a number of compounds. This complexation ability is of particular interest to the food, agrochemical, cosmetic, and pharmaceutical industries for: taste masking, stabilization, increasing solubility, powdering, color masking, and emulsification, to name a few possible uses.
A starch solution can be degraded into cyclodextrin by enzymes known as cyclodextrin glycosyl transferases (1,4-alpha-D-glucan 4-alpha-D-(1,4-alpha-D-glucano)-transferase, E. C. 2.4.1.19), hereinafter termed cyclodextrin glycosyl transferase or CGTase. The CGTase enzymes degrade the liquefied starch by catalyzing cyclization and disproportionation reactions.
Typically, cyclodextrin has been prepared heretofore by variations of the method described by E. B. Tilden and C. S. Hudson (J. American Chemical Society) 64:1432 1942, which method involves treating liquefied starch with a cyclodextrin glycosyl transferase (CGTase) enzyme from Bacillus macetans. All variations of this process have a number of disadvantages. First, since the CGTase is not sufficiently thermostable to be used above the gelatinization temperature of starch, the starch must be pretreated, e.g., with an .alpha.-amylase, to solubilize the starch. It is important that the starch be liquefied to a relatively low DE (Dextrose Equivalent), so after conduct of the starch liquefaction process, the treating agent, normally an .alpha.-amylase, must be inactivated, to obtain good cyclodextrin yield. Second, the Bacillus macerans CGTase is not sufficiently stable to be used at elevated temperatures, and consequently, the enzymatic cyclodextrinization process is carried out at about 50.degree. C., where it is subject to possible microbial contamination. Third, conversion of starch to cyclodextrin (at 50.degree. C., pH 7.0) by the Bacillus macerans CGTase requires extended reaction time, before reasonable yields are achieved.
Importantly, reactions catalyzed by CGTase which cleave the starch molecule can generate a desirable viscosity reduction in liquefied starch solutions by lowering the average molecular weight of the dextrin in the solution (without, in the instance of the CGTase, generating reducing sugars). The CGTase enzymes previously known to the art were produced by such microorganisms as Bacillus macerans, Bacillus circulans, Bacillus stearothermophilus, Bacillus megaterium, Bacillus ohbensis, alkalophilic Bacillus sp., Micrococcus luteus, Micrococcus varians, and Klebsiella pneumoniae. Unfortunately, none of the CGTase enzymes produced by these microorganisms seem to be sufficiently thermostable for use in industrial-scale for a combination of starch liquefaction and cyclodextrin production at temperatures sufficiently elevated to avoid possible microbial contamination.
Enzymatic liquefaction of aqueous starch slurry is widely practiced as the first step in converting starch to dextrose (glucose). To a great extent the starch industry has adopted the liquefaction process of U.S. Pat. No. 3,921,590. Typical conditions are jet cooking at 105.degree. C. for 5 minutes, followed by a 90 minute hold at 95.degree. C., at a starch concentration of 35% DS (dry substance), by weight. The enzyme used in this process is Termamyl.TM. (product of Novo Industri A/S), an .alpha.-amylase from Bacillus licheniformis. Liquefaction is performed at pH about 6.0, followed by saccharification with glucoamylase at a pH of approx 4.5-5.0.
The art has long sought starch liquefaction enzymes capable of liquefying at pH 4.5, in order to eliminate the need for intermediate pH adjustment. .alpha.-amylase from Bacillus stearothermophilus has been suggested for this purpose, but data in this specification show that it does not liquefy well at pH as low as 4.5. U.S. Pat. No. 4,578,532 and U.S. Pat. No. 4,613,570 disclose aciduric .alpha.-amylases from Clostridium, but data in said patents show that their stability at 100.degree. C. or above at pH 4.5 is insufficient.